Sciknow Publications Ltd.

BMB 2013, 1(2): 34-43

Biochemistry & Molecular Biology DOI: 10.12966/bmb.08.02.2013
©Attribution 3.0 Unported (CC BY 3.0)

Balanced Hydrogen Peroxide Metabolism is Central in Controlling

NaCl-Induced Oxidative Stress in Medicinal Legume, Fenugreek
(Trigonella foenum-graecum L.)
Dibyendu Talukdar*
Plant Cell & Stress Biology Lab, Department of Botany, R.P.M. College, University of Calcutta, Uttarpara, West Bengal, India

*Corresponding author (Email: dibyendutalukdar9@gmail.com)

Abstract – Fenugreek, a medicinal and spice-yielding legume, is increasingly facing threats of salinity-induced oxidative stress.
The aim of the present study was to test the antioxidant defense response responsible for H2O2-metabolism in two improved
fenugreek cultivars to prolonged salinity treatment. A pot experiment was designed in controlled environment using 100 mM
NaCl up to 60 days of commencement of treatment (DAS) in a completely randomized block design with four replicates. Foliar
ascorbate (AsA)-redox pool along with activities of superoxide dismutase (SOD), ascorbate peroxidase (APX) and catalases
(CAT) was assayed at 15, 40 and 60 DAS keeping a control in each set. High SOD activity coupled with low ascorbate redox and
declining APX and CAT level in variety Azad led to abnormal rise in H2O2 content at reproductive stage (40 and 60 DAS),
consequently, resulting in NaCl-induced oxidative damage due to high level of lipid peroxidation. Despite initial low H2O2 level,
variety RM-16 experienced onset of oxidative stress at early vegetative stage but remarkably bounced back to normalcy at re-
productive stage of growth exhibiting controlled rise of H2O2 level and decrease in lipid peroxidation as well as electrolyte
leakage. Results revealed H2O2 level was modulated in fine tune by balanced action of SOD and APX, preventing oxidative
damage in RM-16 even under prolonged NaCl-exposure. Enzyme isoforms were involved in regulation of foliar
H2O2-metabolism, the balance of which was found extremely critical in determining NaCl-tolerance of fenugreek genotypes.
Keywords – Ascorbate Redox Pool, Antioxidant Defense, ROS, Enzyme Isoforms, NaCl-tolerance, Growth Stages

ROS scavenging, SOD constitutes the first line of defense, but

1. Introduction
Soil salinity is one of the most severe abiotic stresses affecting
production of the crops worldwide [1]. This problem is more
severe in arid and semi-arid regions, and legume plants al-
ready face a notable impact of salt stress in these regions [2, 3].
The legume family is the second only to the cereals in their
importance to mankind [4], but unfortunately, improvement
of this group of plants for their tolerance against soil salinity
stress have not kept pace with those of cereals and oil seeds.
Salinity induces oxidative stress through the generation of
reactive oxygen species (ROS) within the plant cells [2,3].
The resultant damage is generally manifested by different
alterations at cellular level including membrane lipid perox-
idation and electrolyte leakage, often triggered by over ac-
cumulation of hydrogen peroxide (H2O2)[3]. H2O2 is a highly
diffusible ROS within plant cell and its dual roles as a
stress-inducer and at the same time as a signaling molecule to
up-regulate primary antioxidant defense during oxidative
stress has been increasingly been recognized in different crops
[5-7]. Among the prominent enzymatic systems involved in
it steadily generates H2O2 during dismutation of superoxide
radicals mainly by the action of its membrane bound Cu/Zn
isoforms [8]. This H2O2 is readily scavenged by ascorbate
(AsA) peroxidase (APX) within the AsA–glutathione cycle
and by catalases (CAT) outside this cycle [9]. These three
prominent H2O2 metabolizing enzymes hold key in control-
ling H2O2 level during the onset of salinity-induced oxidative
stress in plants [10].
Trigonella foenum-graecum L. or fenugreek is cultivated
as an annual legume in several Asian, African, North Amer-
ican, and European countries [11], and is extensively used as
spice, leafy vegetable and in medicine as a carminative,
analgesic, anti-inflammatory as well as tonic for gastric
troubles, diabetes, and leucorrhea and as an important source
of steroidal substance, diosgenin for therapeutic uses [12].
However, like many other legumes fenugreek plants face
diverse types of biotic and abiotic stresses like heavy metal,
toxic metalloid arsenic and salinity [11]. Available reports
indicate that fenugreek is moderately salt-tolerant [13], and
the tolerance was reported up to 10 ds m-1salinity level with a
decrease in fresh and dry mass at early growth stage [13]. In a
 Biochemistry & Molecular Biology (2013) 34-43
recent report, Talukdar [14] observed serious decrease in
plant growth parameters and seed yield of fenugreek seedlings
exposed to 100 mM NaCl for 80 days of growth period. De-
spite increasing exposure and sensitivity of fenugreek to sa-
linity, our understanding on the growth of fenugreek plants
under NaCl-induced stress is extremely limited, and no re-
ports are available on antioxidant defense response of this
crop to ROS particularly H2O2 during salinity stress. Due to
extensive use of this legume crop as spices and diverse types
of value-added medicinal/natural product industries, toxicity
of NaCl on its antioxidant metabolism needs urgent study.
The present investigation was, therefore, performed to
investigate NaCl-induced oxidative stress and alteration in
activity of three major H2O2 metabolizing enzymes, SOD,
APX and CAT, as well as AsA redox status in leaves and
roots of NaCl-treated fenugreek plants. In-gel activity of
enzymes was determined by native-PAGE analysis under
control and NaCl-treated conditions.
2. Methods
2.1. Plant materials
The experimental materials comprised of two elite fenugreek
genotypes, namely Azad, and RM-16. These two genotypes
have been widely cultivated in eastern and northern part of
India, and are highly adapted to respective agro-climatic
conditions [15].
2.2. Treatment protocol and plant growth
Dry, healthy and uniform-sized 20 seeds genotype-1 were
surface sterilized in 70% ethanol for 2 min, rinsed twice in
deionized water and then placed on water-moistened filter
papers in 9 cm diameter petridishes in an incubator at 25°C
with 12-hr light following the guidelines of ISTA [16]. Ger-
minated seeds were immediately transferred to twelve inches
earthen pots containing a mixture (total amount of 6.5 Kg) of
this soil, vermiculite and farmyard manure (1:1:1). The ex-
perimental soil was clay loam in texture (clay 32.67%, silt
49.22%, sand 18.11%), neutral (pH 7.0) in reaction and con-
tained 40.7 mg kg-1 exchangeable Na and 7.39 mg kg-1 water
exchangeable Cl. Seedlings were thinned to two per pot after
emergence and watered evenly for their uniform growth until
7 d after first emergence. The pots were kept under control
condition (temperature 20 - 27°C, humidity of 70-77%, neu-
tral light intensity 330-400 μmol m-2 sec-1) during Octo-
ber-December. Salt treatment was commenced on 20-d-old
seedlings. The control plants from each of the four genotypes
were irrigated with distilled water, while others were sub-
jected to salinity stress by watering them with 100 mM NaCl
supplemented distilled water (300 ml water in each pot),
respectively, thrice in a week. Five pots (two plants pot-1)
genotype-1 were arranged in a randomized complete block
design with four replications of each treatment. Salt concen-
tration of 100 mM was found critical in an earlier study for
determining tolerance level of fenugreek genotypes to salt
35
stress [14], and thus, was selected for the present study. Salt
concentration in pot soil was regularly checked by measuring
electrical conductivity with a conductivitimeter (Systronics
M-308, Kolkata, India), and evapotranspirational losses were
compensated daily with deionized water. Leaf samples were
collected at early seedling stage (15 days after commence-
ment of salt treatment or 15 DAS), flowering stage (40 DAS)
and again at harvest (60 DAS) for analysis of different bio-
chemical parameters.
2.3. Estimation of ascorbic acid
Reduced (AsA) and oxidized (DHA) ascorbate contents were
determined by the method of Law et al. [17]. Fresh tissues
(0.5 g) were homogenized in 2.0 ml of K-phosphate buffer
(100 mM, pH 7.0) containing 1 mM EDTA and centrifuged at
10,000 g for 10 min. For the estimation of AsA, 200 μl of
K-phosphate buffer (150 mM, pH 7.4) was added to 750 μl of
the aliquot, while for DHA estimation, 750 μl of the aliquot
was added to 100 μl of dithiotreitol (DTT) followed by vortex
mixing, incubation for 15 min at 20 °C, and addition of 100 μl
of 0.5% (w/v) N’N-ethylmaleimide (NEM). Then, both mi-
crofuge tubes were incubated for 30 s at room temperature. To
each sample tube, 400 μl of 10% (w/v) TCA, 400 μl of H3PO4,
400 μl of 4% (w/v) bipyridyl dye (N’,N-dimethyl bipyridyl),
and 200 μl of 3% (w/v) FeCl3 were added and thoroughly
mixed. The absorbance was recorded at 525 nm after incuba-
tion for 1 h at 37 °C.
2.4. Antioxidant enzyme assay
Fresh leaf tissue (250 mg) was homogenized in 1ml of 50 mM
K-phosphate buffer (pH 7.8) containing 1mM EDTA, 1mM
DTT and 2% (w/v) polyvinyl pyrrolidone (PVP) using chilled
mortar and pestle kept in ice bath. The homogenate was cen-
trifuged at 15,000 g at 4 °C for 30 min. Clear supernatant was
used for enzyme assays. For measuring APX activity, the
tissue was separately ground in homogenizing medium con-
taining 2.0 mM ascorbate in addition to the other ingredients.
All assays were done at 25 °C with at least four replicates.
Soluble protein content was determined according to Brad-
ford [18] using bovine serum albumin as a standard.
SOD (EC 1.15.1.1) activity was determined by nitro blue
tetrazolium (NBT) photochemical assay, according to Beau-
champ and Fridovich [19]. In this method 1ml of solution
containing 50 mM K-phosphate buffer (pH 7.8), 9.9 mM
L-methionine, 57 µM NBT, 0.025% triton-X-100 was added
into small glass tubes, followed by 20 µl of enzyme extract.
Reaction was started by adding 10 µl of riboflavin solution
(0.044 mg/ml) and the absorbance of solution was measured
at 560 nm. SOD activity was expressed as U (unit) mg-1 pro-
tein. One unit of SOD was equal to that amount which causes
a 50% decrease of SOD-inhibited NBT reduction. SOD iso-
zymes were individualized by native-PAGE on 10% acryla-
mide gels and were localized by a photochemical method [19].
Activity staining gels were incubated for 30 min in 50 mM
K-phosphate buffer at pH 7.5 containing 2 mM KCN or 5 mM
H2O2. Cu/Zn-SODs are inhibited by KCN and H2O2;
36 Dibyendu Talukdar： Balanced Hydrogen Peroxide Metabolism is Central in Controlling NaCl-Induced Oxidative Stress
in Medicinal Legume, Fenugreek (Trigonella foenum-graecum L.)
Fe-SODs are inactivated by H2O2 but resistant to KCN and
Mn-SODs are resistant to both inhibitors.
APX (EC 1.11.1.11) activity was assayed following me-
thods of Nakano and Asada [20]. Three milliliters of the
reaction mixture contained 50 mM K-phosphate buffer (pH
7.0), 0.1 mM EDTA, 0.5 mM AsA, 0.1 mM H2O2 and 0.1 ml
enzyme extract. The H2O2-dependent oxidation of AsA was
followed by a decrease in the absorbance at 290 nm (ε = 2.8
mM-1cm-1). APX activity was expressed as nmol AsA oxi-
dized min-1mg-1 protein. Native-PAGE of APX isozymes
were performed in 4 % gel and stained following Mittler and
Zilinskas [21] based on the inhibition of NBT reduction by
AsA.
CAT (EC 1.11.1.6) activity was measured following the
procedure of Aebi [22]. CAT was extracted in 50 mM
K-phosphate buffer (pH 7.0) and 0.5 % PVP, and its activity
was assayed by measuring the reduction of H2O2 at 240 nm (ε
= 39.4 M-1 cm-1) for 1 min [26]. CAT isozyme profiling was
performed on 6% acrylamide gels and the activity was loca-
lized in the gels following Woodbury et al. [23].
2.5. Hydrogen peroxide estimation
The H2O2 content of fully matured leaves will be colorimetr-
ically measured as described by Mukherjee and Choudhuri
[24] by extracting leaf samples in cold acetone and mixing of
an aliquot (3 ml) of the extracted solution with 1 ml of 0.1%
titanium dioxide in 20% (v:v) H2SO4. The concentration of
H2O2 would be calculated from a standard curve.
2.6. Estimation of lipid peroxidation
Lipid peroxidation rates were determined by measuring the
malondialdehyde (MDA) equivalents following the method of
Hodges et al. [25]. About 0.5 g of fresh tissue was homoge-
nized in a mortar with 80 % ethanol. The homogenate was
centrifuged at 3,000 g for 12 min at 4 °C. The pellet was
extracted twice with the same solvent. The supernatants were
pooled and 1 ml of this sample was added to a test tube with
an equal volume of either the solution comprised of 20 %
TCA and 0.01 % butylated hydroxy toluene (BHT) or solution
of 20 % TCA, 0.01 % BHT and 0.65 % TBA. Samples were
heated at 95 °C for 25 min and cooled to room temperature.
Absorbance was measured at 450, 532 and 600 nm. Level of
lipid peroxides was calculated following Hodges et al. [25]
and expressed as nmol MDA g−1 FW.
2.7. Assay of Electrolyte leakage
Electrolyte leakage (EL) was assayed by measuring the ions
leaching from tissue into deionised water [26]. Fresh samples
(100 mg) were cut into small pieces (about 5 mm segments)
and placed in test tubes containing 10 ml deionised water.
Tubes were kept in a water bath at 32 °C for 2 h. After incu-
bation, electrical conductivity (EC1) of the bathing solution
was recorded with an electrical conductivity meter (Systron-
ics M-308, Kolkata, India). The samples were then autoclaved
at 121 °C for 20 min to completely kill the tissues and release
all electrolytes. Samples were then cooled to 25 °C and final
electrical conductivity (EC2) was determined. The EL was
expressed as a percentage by the formula, (EL%) = (EC1) /
(EC2) ×100.
2.8. Statistical analysis
The results presented here are the mean values ± standard
error (SE) of at least four replicates. Means were compared by
ANOVA using the SPSS v. 10 (SPS Inc, USA) and evaluated
using Duncan’s Multiple Range Test at P ≤ 0.05.
3. Results
3.1. NaCl-induced H2O2 accumulation, lipid peroxidation
and electrolyte leakage (EL%)
At the start of the treatment (0 DAS), no significant variation
was observed among the genotypes for foliar H2O2, MDA and
EL%. However, compared to control plants, accumulation of
H2O2 and MDA was significantly lower in variety Azad at 15
DAS (Fig. 1a, b). Both the parameters were markedly higher
than control at 40 DAS and then became stable (Fig. 1a, b).
The increase of H2O2 was 3.5 fold while MDA level was
increased by about 3-fold in Azad at 40 DAS. Statistically
significant rise was also observed for EL% in this variety at 40
DAS and 60 DAS (Fig. 1c). By contrast, H2O2 content was
markedly low (nearly 1.8-fold) but MDA content and EL%
was quite high (4-5-fold) in RM-16 at 15 DAS in relation to
control (Fig. 1a-c). At 40 DAS, H2O2-level was increased by
about 3-fold in RM-16 from the level recorded at 15 DAS, and
was marginally changed at 60 DAS. MDA content and EL%,
however, were normal in this genotype at 40 DAS and also, at
60 DAS (Fig. 1b, c).
 Biochemistry & Molecular Biology (2013) 34-43 37

Fig. 1. Changes in (a) H2O2 content, (b) malondealdehyde (MDA), and (c) electrolyte leakage % in leaves of fenugreek va-
riety Azad and RM-16 along with a control at initiation of 100 mM NaCl treatment (0) and at 15, 40 and 60 days after com-
mencement (DAS) of salt treatment. Data presented here are means ±SE of four replicates. Means with different small letters are
significantly different at P ≤ 0.05 by ANOVA followed by Duncan’s Multiple Range Test.
Among the H2O2-metabolizing enzymes, statistically sig-
3.2. Antioxidant defense responses to NaCl-treatment nificant lower SOD activity compared to control was ob-
Foliar AsA content was quite normal in variety Azad at 15 served in Azad seedlings since 15 DAS but the trend was
DAS. As the treatment progressed further, AsA level and its reversed at 40 DAS and 60 DAS exhibiting a rise in enzyme
redox state (AsA/AsA+DHA, DHA-dehydroascorbate) was activity over control by about 3-fold and 5-fold, respectively
declined by about 2-fold in this variety at 40 DAS and was (Table 1). SOD activity was also low in RM-16 (3-fold) at 15
further reduced at 60 DAS (Table 1). Contrastingly, in variety DAS. However, it was increased by about 2-fold over control
RM-16, AsA content and its redox state were significantly low at 40 DAS and marginally varied at 60 DAS (Table 1). APX
at 15 DAS but were enhanced approximately 2-fold at 40 and CAT activity was quite normal in Azad at 15 DAS, but
DAS. High AsA redox was maintained in this variety at 60 was declined significantly at 40 DAS and 60 DAS (Table 1).
DAS, also (Table 1).
38 Dibyendu Talukdar： Balanced Hydrogen Peroxide Metabolism is Central in Controlling NaCl-Induced Oxidative Stress
in Medicinal Legume, Fenugreek (Trigonella foenum-graecum L.)

By contrast, APX activity was normal while very high CAT continued at 60 DAS, also. CAT level was significantly lower
activity (about 6-fold) was recorded in variety RM-16 at 15 than control at 40 DAS and 60 DAS in salt-treated RM-16
DAS. At 40 DAS, APX level began to rise and the trend was variety (Table 1).

Table 1. Effect of 100 mM NaCl treatment on reduced (AsA), redox state of ascorbate and activities of superoxide dismutase
(SOD, U min-1 mg-1 protein), ascorbate peroxidase (APX, nmol AsA oxidized min−1 mg−1 protein), and catalases (CAT, nmol
H2O2 degraded min-1 mg-1 protein) in leaves of control and NaCl-treated fenugreek (Trigonella foenum-graecum L.) variety
Azad and RM-16 during initiation of treatment (0) and at 15, 40 and 60 days after commencement of treatment (DAS)

Parameters DAS Control Azad RM-16

-1
AsA (µmol g FW) 0 1.73 ±1.04a a' 1.75 ±1.06a a' 1.69 ±1.24a b'
15 1.79 ±1.48a a' 0.88 ±0.10c b' 0.99 ±1.73b c'
40 1.70 ±0.73b a' 0.62 ±0.13c c' 3.26 ±0.88a a'
60 1.71 ±0.79b a' 0.48 ±0.10c d' 3.33 ±0.79a a'
AsA redox [AsA/
0 0.856 ±0.09a a' 0.891 ±0.09a a' 0.880 ±0.08a a'
(AsA+DHA)]
15 0.832 ±0.11a a' 0.453 ±0.10c b' 0.677 ±0.12b b'
40 0.842 ±0.08a a' 0.351 ±0.09b c' 0.868 ±0.09a a'
60 0.861 ±0.09b a' 0.289 ±0.11c d' 0.905 ±0.09a a'
SOD 0 61.5 ±5.1a a' 68.9 ±4.9a c' 73.3 ±4.7a b'
15 60.9 ±5.8a a' 39.8 ±4.8b d' 24.7 ±5.1c c'
40 58.8 ±4.8c a' 204.8 ±10.5a b' 146.6 ±9.8b a'
60 62.6 ±11.6c a' 345.6 ±9.8a a' 151.1 ±6.9b a'
APX 0 65.8 ±7.8a a' 69.9 ±8.1a a' 70.5 ±8.9a c'
15 74.7 ±8.6a a' 70.7 ±12.6a a' 69.3 ±4.3a c'
40 77.9 ±4.9b a' 45.6 ±6.3c b' 120.4 ±18.5a b'
60 59.7 ±6.1b a' 31.3 ±5.5c c' 134.5 ±11.3a a'
CAT 0 29.1 ±4.7b a' 30.1 ±6.7b a' 49.7 ±5.1a b'
15 30.5 ±3.8b a' 30.8 ±6.4b a' 81.6 ±4.9a a'
40 30.1 ±4.1a a' 17.3 ±4.7c b' 21.5 ±5.3b c'
60 28.7 ±5.0a a' 16.9 ±5.1b b' 18.6 ±3.1b c'

Data were presented as means ± SE of four replicates. Different small letters in each row indicate significant differences
among genotypes and letters with prime in each column indicate significant differences among treatments (for a particular trait)
at P ≤ 0.05 by ANOVA followed by Duncan’s Multiple Range Test.
could be resolved in variety RM-16 (Fig. 2 a). Occurrence of
3.3. In-gel activity of antioxidant enzymes under Mn SOD isoforms was confirmed by inhibitor (H2O2 and
As-exposure
KCN) study (Fig. 2b). Like control plants, a total of three
Altogether three activity bands were resolved for SOD on the isoforms (APX 1, APX 2, and APX 3) were consistently
basis of their increasing mobility and sensitivity to H2O2 and resolved in the leaf extract of variety Azad but with lower
KCN, and zymograms obtained at 60 DAS was presented (Fig. intensity in APX 2 and 3 at 60 DAS in comparison to control
2a, b). Two Cu/Zn isoforms (I and II) were consistently re- (Fig. 3a). In salt-treated RM-16, all the three isoforms were
solved by native PAGE in the leaf extract of control plants resolved as strong band at 60 DAS (Fig. 3A). CAT activity
(Fig. 2a). At 15 DAS, Cu/Zn-SOD I and II were visualized in was uniformly resolved as a single zone across the genotypes,
leaves of two genotypes but with much lower intensity in both exhibiting lower band intensity in both varieties at 60 DAS in
Azad and RM-16 (Figure not shown). At 60 DAS, in addition comparison to that in control (Fig. 3b).
to Cu/Zn I and II, two Mn SOD isoforms (Mn SOD I and II)
was distinctly visualized in variety Azad but one Mn SOD I
 Biochemistry & Molecular Biology (2013) 34-43 39

Fig. 2. (a) Activity gel of superoxide dismutase (SOD) in

native-PAGE on 10% acrylamide gels of leaf extracts of two
fenugreek (Trigonella foenum-graecum L.) genotypes at 60
days after commencement of 100 mM NaCl treatment; Lane
1-control, lane 2 and 3- variety Azad, lane 4 and 5- RM-16,
and (b) inhibitor studies with H2O2 and KCN and visualiza-
tion of SOD isoforms in native PAGE of leaf extracts of fe-
nugreek plants; lanes 1 and 2- control, lanes 3 and 4- variety 4. Discussion
Azad, and lanes 5 and 6- RM-16 at 60 days after com-
Salinity is known to induce oxidative stress in plants by ge-
mencement of salt treatment. The control plants containing
nerating various types of ROS [2, 3], resulting in arrays of
only Cu/Zn SOD I and II showed no bands in inhibitor study.
responses in plants, including growth inhibition and read-
justment of antioxidant metabolism [3]. ROS, such as H2O2
Fig. 3. Effects of 100 mM NaCl on isozymes banding of
and superoxide radicals (O2-) are central components of the
(a) APX activity in native PAGE on 4% acrylamide gels of
signal transduction cascade involved in plant adaptation to the
leaf extracts of two fenugreek (Trigonella foenum-graecum L.)
abiotic stress [5, 6]. H2O2 is a highly diffusible ROS across
genotypes at 60 days after commencement of treatment; lane
cellular membranes and inflicts oxidative damage to
1 and 2- control plant, lanes 3 and 4- variety Azad, lanes 5 and
thiol-containing enzymes [7, 27]. On the other hand, it can
6- variety RM-16, and (b) CAT activity in native PAGE on
induce antioxidant defense to be up-regulated against oxida-
6% acrylamide gels of leaf extracts of two fenugreek (Tri-
tive stress as a signaling molecule [7]. Therefore, regulation
gonella foenum-graecum L.) genotypes at 60 days after
of its concentration at a particular level within cellular envi-
commencement of treatment; lane 1and 2- control plants,
ronment is the most vital during plant growth and develop-
lanes 3 and 4- variety Azad, and lanes 5 and 6- variety RM-16.
ment [5-7, 27, 28]. To ascertain the dual roles, exacerbating
damage or signaling the activation of defense response, and
cellular control of H2O2 under NaCl exposure, activities of
SOD, APX and CAT along with total and redox state of AsA
40 Dibyendu Talukdar： Balanced Hydrogen Peroxide Metabolism is Central in Controlling NaCl-Induced Oxidative Stress
in Medicinal Legume, Fenugreek (Trigonella foenum-graecum L.)
were assessed in fenugreek plants exposed to 100 mM NaCl
for 60 days of growth period as a long-term salt treatment.
The response of antioxidant defense to NaCl treatment in two
widely cultivated varieties of fenugreek in the present study is
not straightforward. Time-bound measurements at 15, 40 and
60 DAS revealed normal level of H2O2 at 15 DAS but its huge
increase at 60 DAS in leaves of both varieties. The rise of
H2O2 in variety Azad was associated with significant en-
hancement in MDA and EL %, marking the onset of
NaCl-induced oxidative damage in the photosynthetic organ
of variety Azad. MDA is a cytotoxic aldehyde, produced from
lipid peroxidation of membrane due to excessive ROS ac-
cumulation during oxidative stress [2, 3, 28]. This along with
high level of H2O2 is often considered as biochemical markers
of oxidative stress in crop plants [2, 29, 30]. In salini-
ty-induced genotypes of pea, grass pea and chickpea, rise in
H2O2 coupled with abnormal accumulation of MDA resulted
in significant damage in structural integrity of membrane in
NaCl-sensitive genotypes, as was evidenced by high EL% [2,
31, 32]. The positive relationship between over-accumulation
of oxidative species, lipid peroxidation and increased mem-
brane leakage were also observed during heavy metal toxicity
and arsenic-induced oxidative damage of fenugreek, common
beans, lentil and other crop plants [30, 33, 34], resulting in
severe impediment in growth potential and grain yield in
sensitive genotypes [2, 35], and also during strong
weed-induced phytotoxicity on vital cellular processes of
grass pea and lentil plants [36, 37]. This situation, however,
was not true in case of present RM-16 seedlings exposed to
NaCl treatment. A dissection through growth stages revealed
that rise in foliar H2O2 level in this variety at 40 and 60 DAS
was inversely correlated with MDA content (r = -0.797, n
=10). Similarly, MDA content was very high in this genotype
at 15 DAS when H2O2 level was significantly lower even than
control plants. The results strongly suggested that variety
RM-16 experienced oxidative damage at early vegetative
stage but remarkably enough, it withstand negative impact of
NaCl-stress at flowering (40 DAS) and pod-bearing stages
(60 DAS). Similar situation was encountered in salt-stressed
genotypes of grass pea including several novel dwarf mutants
and genetic stocks, differing in growth pattern and salinity
tolerance [38-41] and was explained as manifestation of salt
tolerance due to overhauling of intrinsic biochemical defense
mechanism during plants’ exposure to salinity [42, 43].
This completely reverse situation between two varieties in
identical experimental protocol can be better explained if we
consider enzymatic regulation of H2O2 in the treated seedlings.
High SOD activity strongly suggested NaCl-induced genera-
tion of excess superoxide radicals and subsequently, their
dismutation formed huge H2O2 in treated seedlings during 40
and 60 DAS. To counterbalance this situation, plants have
well-integrated H2O2-detoxification system [9, 10, 20]. With
several isozymes, APX is the most prolific H2O2-scavenging
enzyme within AsA-GSH cycle but it exclusively requires
AsA as its co-factor during scavenging [9, 20]. On the other
hand, CAT can decompose H2O2 without consuming any
reducing power outside this cycle [10]. In variety Azad, de-
clining APX and CAT level at reproductive growth stages,
presumably, jeopardized the H2O2-scavenging capacity of the
genotypes, resulting in abnormal rise in H2O2 level under
long-term salt exposure. Declining level of APX might be due
to marked decrease in AsA level and its redox state in Azad at
this treatment period. Besides serving as co-factor of APX,
AsA itself can detoxify ROS through non-enzymatic me-
chanism [44] and its availability and redox state plays key
roles in signaling network through controlled metabolism of
H2O2 during oxidative stress [7]. Intracellular re-distribution
of antioxidant defense in response to toxic level of H2O2 under
NaCl exposure was nicely demonstrated in different aneup-
loids as well as in a novel ascorbate-deficient mutant [41, 45]
and also in model plant Arabidopsis thaliana [46]. Prolonged
NaCl-exposure, presumably led to crippling of AsA-mediated
antioxidant defense in variety Azad, resulting in significant
rise of H2O2 content under NaCl-treatment. This situation,
however, got a significant turn in variety RM-16. Powered by
normal APX level, AsA-redox and high CAT activity, the
genotype was able to reduce H2O2 level even below control
level during 15 DAS but this could not prevent the severe
oxidative damage the genotype experienced at early seedling
stage. Remarkable recovery of this genotype manifested at its
reproductive stage (40 and 60 DAS) by normal MDA and
EL% was obviously possible through modulation of antioxi-
dant defense in response to prolonged exposure to NaCl.
Considerable rise of SOD activity coupled with low CAT
level might be instrumental to generate and accumulate H2O2
in a level higher than control but significantly this was not so
high when compared with Azad at the same growth period.
Obviously, the role of APX in this context is worth men-
tioning. High APX level coupled with elevated AsA redox
pool in salt-treated RM-16 during 40 and 60 DAS ensured
efficient scavenging of H2O2, maintaining it a particular level
that was higher than control but was not toxic to plant growth
and development even under prolonged salt exposure. Pre-
sumably, leaf H2O2 concentration in this genotype was regu-
lated in controlled way by opposite mechanisms of prominent
H2O2-metabolizing enzymes, balancing it to a particular level
during later stages of growth. Certainly, H2O2 played dual
roles in stress perception of NaCl-treated two fenugreek ge-
notypes in a concentration-dependent manner; it promoted
plant growth of RM-16 at 40 and 60 DAS at a level which was
higher than control but lower to variety Azad, and at the same
time, escalated NaCl-induced oxidative damage in Azad in a
concentration that was far higher than control. Obviously,
H2O2 level in latter case reached its toxic concentration which
inflicted damage but was regulated in RM-16, favoring plant
growth under NaCl treatment. In cadmium-treated lentil
crops primed with calcium, enhanced H2O2 promoted plant
growth through modulation of antioxidant defense compo-
nents [47]. The functions and implications of H2O2 as a
stressor or as a signaling molecule were also studied in Bras-
 Biochemistry & Molecular Biology (2013) 34-43
sica oleracea roots, subjected to both short and long salt stress,
revealing accumulation of H2O2 was accompanied with re-
covery of APX and CAT activity under long salt stress [48].
Certainly, the status of cellular H2O2 informed the plant cell
about the severity of the oxidative stress and hence the ap-
propriate level of antioxidant enzyme activities through in-
duction [47-50]. Present results indicates that the H2O2 level is
highly critical in stress perception, which can be regu-
lated/adjusted in favor of plant growth and yield perfor-
mances during prolonged exposure to high NaCl. This critical
balance, however, was lost in variety Azad during prolonged
NaCl-exposure, leading to un-regulated generation and ac-
cumulation of H2O2 with concomitant induction of oxidative
stress in its severest form. Thus, it seems clear that oxidative
damage at initial growth stages was effectively prevented in
fenugreek variety RM-16 by mitigating membrane damage at
reproductive stages, but it was quite impossible for the Azad
to recover from NaCl-induced oxidative damage due to col-
lapse of its H2O2-scavenging machinery and continuous rise
in SOD activity in response to long-term salt treatment.
Changes in antioxidant enzyme activities coincided with a
variable increase or decrease of their individual isoform ex-
pression. Analysis of isoforms and inhibitor studies in leaf
extracts of both varieties at native PAGE revealed that the
increase in SOD activity in Azad at 60 DAS over control was
due to over-activity of both Cu/Zn I and II isoforms, as sug-
gested by high staining intensity, and origin of two new iso-
forms resolved as Mn SOD I and II. On the other hand, sig-
nificant enhancement in SOD activity in variety RM-16 at 60
DAS might be conferred by a new isoform Mn SOD I along
with elevated expression of Cu/Zn SOD I and II. The supply
of NaCl reportedly enhanced the activity of Cu/Zn-SOD in
wheat seedlings [51, 52]. Present result revealed origin of two
novel isoforms as two Mn-SODs (I and II) in Azad and one
Mn SOD I in variety RM-16. It seems likely that increased
activity of SOD at 15 DAS was mainly mediated through
existing isoforms but as the treatment progressed induction of
SOD expression was required in different cellular compart-
ments to combat the elevated generation of superoxide radi-
cals due to NaCl-exposure. One of the main ROS sources in
the cell is the photosynthesis process [10]. While Cu/Zn iso-
forms are predominantly present in chloroplast and cytosol,
Mn-SODs are located in peroxisomes and mitochondria [8],
suggesting participation of SOD isoforms in different cellular
compartments due to NaCl-treatment in the present material.
For APX, the increased activity in RM-16 at 60 DAS was
mainly due to enhanced expression of APX 1, APX 2 and
APX 3 isozymes, as was evidenced by their intense bands in
zymograms. By contrast, the declining activity in Azad at 60
DAS was manifested by diminishing intensity of APX bands,
particularly APX 2 and APX 3. Leaf APX isoforms are ra-
pidly deactivated by over-accumulating H2O2 at low AsA
pool [53], and this was, perhaps, one of the prime reasons for
reduced APX level in NaCl-treated variety Azad at later stages
of growth. Origin of new isoforms of SOD and APX and
enhanced expression of existing isoforms was also observed
41
in fenugreek plants, exposed to long-term arsenic exposure
[33]. High CAT activity in RM-16 at 15 DAS but its low
activity in both varieties at 60 DAS were manifested as
change in intensity of bands in zymogram and suggested
down-regulation of its expression during prolonged
salt-exposure, similar to the findings in different crop and
medicinal plants under salt-stress [54]. Induction of new SOD
isoforms at 60 DAS has immense significance as the plants
were then at vital reproductive stages and major changes
(increase or decrease) in H2O2 level occurred in this period.
Present results clearly indicated criticality of reproductive
period in determining regulation of H2O2-metabolism through
NaCl-induced enzyme expressions. The variations in isozyme
pattern and their correspondence to total assayed activity
suggested that the three H2O2-metabolizing enzymes re-
sponded strongly to NaCl-induced oxidative stress in fenu-
greek plants.
5. Conclusion
For the first time, effect of prolonged NaCl-treatment was
studied in two fenugreek genotypes. Results revealed signif-
icant genotypic differences in response to 100 mM NaCl
treatment and even differences between growth stages of a
particular genotype. NaCl treatment induced changes in H2O2
metabolism by modulating antioxidant defense components in
differential manners in two genotypes. Results revealed low
H2O2 in RM-16 failed to prevent onset of oxidative stress at
initial growth period but its rise was regulated by balanced
action of SOD and APX during vital flowering and
pod-bearing stages to restore normal antioxidant metabolism,
as was evident from normal MDA and EL% at reproductive
growth stage. By contrast, the negative impact of NaCl ex-
posure was manifested in Azad due to severe perturbation in
H2O2-metabolism by aberrant responses from SOD, APX and
CAT, the severity of which was increased at later stages of
growth. Present result revealed that H2O2-metabolism holds
the key in determining tolerance of two fenugreek genotypes
to prolonged exposure of NaCl-induced oxidative stress, and
APX rather than CAT played pivotal role in balancing H2O2
level in the backdrop of high SOD activity during progression
of stress period. Understanding the intrinsic modulation of
physiological and antioxidant metabolism by H2O2 through
different growth stages would be an important step in for-
mulating effective breeding strategies for improving
NaCl-tolerance in crop plants.
Acknowledgments
Author is thankful to University Grants Commission, India
for granting financial assistance in the form of research
project (Grant no. PSW-047/11-12 ERO) to carry out present
work.
42 Dibyendu Talukdar： Balanced Hydrogen Peroxide Metabolism is Central in Controlling NaCl-Induced Oxidative Stress
in Medicinal Legume, Fenugreek (Trigonella foenum-graecum L.)
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